The 90-kDa heat shock protein (Hsp90) assists in the proper folding of numerous mutated or overexpressed signal transduction proteins that are involved in cancer. Consequently, there is considerable interest in developing chemotherapeutic drugs that specifically disrupt the function of Hsp90. Here, we investigated the extent to which a novel novobiocin-derived C-terminal Hsp90 inhibitor, designated KU135, induced antiproliferative effects in Jurkat T-lymphocytes. The results indicated that KU135 bound directly to Hsp90, caused the degradation of known Hsp90 client proteins, and induced more potent antiproliferative effects than the established N-terminal Hsp90 inhibitor 17-allylamino-demethoxygeldanamycin (17-AAG). Closer examination of the cellular response to KU135 and 17-AAG revealed that only 17-AAG induced a strong up-regulation of Hsp70 and Hsp90. In addition, KU135 caused wild-type cells to undergo G 2 /M arrest, whereas cells treated with 17-AAG accumulated in G 1 . Furthermore, KU135 but not 17-AAG was found to be a potent inducer of mitochondria-mediated apoptosis as evidenced, in part, by the fact that cell death was inhibited to a similar extent by Bcl-2/Bcl-x L overexpression or the depletion of apoptotic protease-activating factor-1 (Apaf-1). Together, these data suggest that KU135 inhibits cell proliferation by regulating signaling pathways that are mechanistically different from those targeted by 17-AAG and as such represents a novel opportunity for Hsp90 inhibition.

BackgroundAppropriate responses to damaged DNA are indispensible for preserving genome stability and preventing cancer. Tumor viruses often target DNA repair machinery to achieve transformation. The Human T-cell leukemia virus type I (HTLV-I) is the only known transforming human retrovirus and the etiological agent of Adult T-cell Leukemia (ATLL). Although HTLV-I-transformed leukemic cells have numerous genetic lesions, the precise role of the viral tax gene in this process is not fully understood.ResultsOur results show a novel function of HTLV-I oncoprotein Tax as an inducer of genomic DNA double strand breaks (DDSB) during DNA replication. We also found that Tax acts as a potent inhibitor of homologous recombination (HR) DNA repair through the activation of the NF-kB pathway. These results were confirmed using HTLV-I molecular clones expressing Tax at physiological levels in a natural context. We further found that HTLV-I- and Tax-transformed cells are not more susceptible to DNA damaging agents and repair DNA lesions at a rate similar to that of normal cells. Finally, we demonstrated that during S phase, Tax-associated DDSB are preferentially repaired using the error-prone non-homologous end joining (NHEJ) pathway.ConclusionsThis study provides new insights in Tax effects on DNA repair and genome instability. Although it may not be self sufficient, the creation of DNA breaks and subsequent abnormal use of the non-conservative NHEJ DNA repair during the S phase in HTLV-I-infected Tax-expressing cells may cooperate with other factors to increase genetic and genome instability and favor transformation.

Mitochondrial outer membrane permeabilization and the release of intermembrane space proteins, such as cytochrome c, are early events during intrinsic (mitochondria-mediated) apoptotic signaling. Although this process is generally accepted to require the activation of Bak or Bax, the underlying mechanism responsible for their activation during true intrinsic apoptosis is not well understood. In the current study, we investigated the molecular requirements necessary for Bak activation using distinct clones of Bax-deficient Jurkat T-lymphocytes in which the intrinsic pathway had been inhibited. Cells stably overexpressing Bcl-2/Bcl-x L or stably depleted of Apaf-1 were equally resistant to apoptosis induced by the DNA-damaging anticancer drug etoposide as determined by phosphatidylserine externalization and caspase activation. Strikingly, characterization of mitochondrial apoptotic events in all three drug-resistant cell lines revealed that, without exception, resistance to apoptosis was associated with an absence of Bak activation, cytochrome c release, and mitochondrial membrane depolarization. Furthermore, we found that etoposide-induced apoptosis and mitochondrial events were inhibited in cells stably overexpressing either full-length X-linked inhibitor of apoptosis protein (XIAP) or the BIR1/BIR2 domains of XIAP. Combined, our findings suggest that caspase-mediated positive amplification of initial mitochondrial changes can determine the threshold for irreversible activation of the intrinsic apoptotic pathway.Apoptosis is a gene-regulated form of cell death that is critical for normal development and tissue homeostasis. Disruptions in the control of apoptosis can contribute to the onset of various pathological states including cancer, where avoidance of apoptosis confers a survival advantage to tumorigenic cells. Apoptosis is mediated by a family of cysteine proteases that cleave after aspartate residues (caspases) and can be activated by two distinct signaling pathways.The intrinsic (mitochondria-mediated) pathway is activated by cytotoxic stressors, such as DNA damage, ␥-radiation, growth factor withdrawal, and heat. Such stimuli are known to cause mitochondrial outer membrane permeabilization (MOMP) 2 and stimulate the release of cytochrome c, second mitochondria-derived activator of caspase (Smac, also known as DIABLO), and Omi (also known as HtrA2) into the cytosol, where they work together to activate the initiator procaspase-9 within the apoptotic protease-activating factor-1 (Apaf-1) apoptosome complex (1). Once activated, caspase-9 activates effector procaspase-3 or -7, which, in turn, can cleave various protein substrates, leading to the morphological and biochemical features of apoptosis.The process of MOMP is generally thought to require the activation of a multidomain Bcl-2 family protein, notably Bax or Bak (2, 3). Cells deficient in either Bax or Bak display relatively minor defects in apoptosis, whereas doubly deficient cells are often found to be highly resistant to mitochondria-mediated ap...

Exposure of cells to hyperthermia is known to induce apoptosis, although the underlying mechanisms are only partially understood. Here, we examine the molecular requirements necessary for heat-induced apoptosis using genetically modified Jurkat T-lymphocytes. Cells stably overexpressing Bcl-2/ Bcl-x L or stably depleted of Apaf-1 were completely resistant to heat-induced apoptosis, implicating the involvement of the mitochondria-mediated pathway. Pretreatment of wild-type cells with the cell-permeable biotinylated general caspase inhibitor b-VAD-fmk (biotin-Val-Ala-Asp(OMe)-CH 2 F) both inhibited heat-induced apoptosis and affinity-labeled activated initiator caspase-2, -8, and -9. Despite this finding, however, cells engineered to be deficient in caspase-8, caspase-2, or the caspase-2 adaptor protein RAIDD (receptor-interacting protein (RIP)-associated Ich-1/CED homologous protein with death domain) remained susceptible to heat-induced apoptosis. Additionally, b-VAD-fmk failed to label any activated initiator caspase in Apaf-1-deficient cells exposed to hyperthermia. Cells lacking Apaf-1 or the pro-apoptotic BH3-only protein Bid exhibited lower levels of heat-induced Bak activation, cytochrome c release, and loss of mitochondrial membrane potential, although cleavage of Bid to truncated Bid (tBid) occurred downstream of caspase-9 activation. Combined, the data suggest that caspase-9 is the critical initiator caspase activated during heat-induced apoptosis and that tBid may function to promote cytochrome c release during this process as part of a feed-forward amplification loop.Sublethal heat exposure is known to induce an evolutionarily conserved adaptive response known as the heat shock response. A key feature of this response includes the transcriptional up-regulation of several heat shock proteins that are known to confer protection against a subsequent exposure to an otherwise lethal cellular stressor, including ␥-radiation, hyperthermia, and chemotherapeutic agents (1, 2). By comparison, an initial exposure of cells to a more severe or prolonged bout of hyperthermia is known to overcome this protective heat shock response and induce apoptosis or necrosis (3). Significantly, because hyperthermia can induce apoptosis, it is currently being tested in combination with conventional anti-cancer therapy in clinical trials for advanced malignancies (4, 5). Additionally, there is a growing interest in developing ways to more selectively target tumor cells with hyperthermia for therapeutic use (6, 7).There are two distinct apoptotic pathways: (i) the mitochondria-mediated (i.e. intrinsic) pathway and (ii) the receptor-mediated (i.e. extrinsic) pathway. The extrinsic pathway is activated upon binding of a death ligand to its cognate receptor (e.g. Fas binding to the Fas receptor), which causes the receptors to move within close proximity to one another and recruit the adaptor protein Fas-associated protein with death domain (FADD), followed by the recruitment of initiator procaspase-8 or -10 (8, 9). This protein comp...